The present invention relates to positive response sensors and, particularly, to enzymatic biosensors in which two reaction schemes provide a positive response.
There are many types of sensors designed to detect the presence of chemical species, for example, on surfaces or within solutions. Such sensors exhibit signals based on a wide variety of chemical, electrical, or physical responses. Many such sensors are based upon xe2x80x9cnegative responsesxe2x80x9d. In negative response sensors, the chemical analyte of interest inhibits or retards a chemical or physical process that would otherwise take place within the sensor in the analyte""s absence. The term xe2x80x9cnegative response sensorxe2x80x9d thus generally refers sensors in which the presence of a target analyte results in the absence of or the reduction of a signal change or a signal change.
Enzymatic proteins are remarkable natural catalysts in that they selectively catalyze many reactions under relatively mild reaction conditions. Enzymes also offer the potential to perform sterio- and regio-selective reactions not readily accomplished with conventional chemistry. As used herein, the term xe2x80x9cenzymexe2x80x9d refers generally to proteins that catalyze biochemical reactions. These xe2x80x9cbiopolymersxe2x80x9d include amide-linked amino acids and typically have molecular weights of 5,000 or greater. A compound for which a particular enzyme catalyzes a reaction is typically referred to as a xe2x80x9csubstratexe2x80x9d of the enzyme.
In general, six classes or types of enzymes (as classified by the type of reaction that is catalyzed) are recognized. Enzymes catalyzing reduction/oxidation or redox reactions are referred to generally as EC 1 (Enzyme Class 1) Oxidoreductases. Enzymes catalyzing the transfer of specific radicals or groups are referred to generally as EC 2 Transferases. Enzymes catalyzing hydrolysis are referred to generally as EC 3 hydrolases. Enzymes catalyzing removal from or addition to a substrate of specific chemical groups are referred to generally as EC 4 Lyases. Enzymes catalyzing isomeration are referred to generally as EC 5 Isomerases. Enzymes catalyzing combination or binding together of substrate units are referred to generally as EC 6 Ligases.
Enzymes have been known since the early 1960""s to be useful tools for detecting the presence of chemical species. Rogers, K. R., Biosensors Bioelectronics, 10, 533 (1995). A number of enzymatic biosensors have been designed to detect a variety of different compounds including, for example, glucose, creatinine, urea, and cholinesterase inhibitors. Parente, A. H., Marques, E. T. Jr., Appl. Biochem. Biotechnol. 37, 3, 267 (1992); Yang, S., Atanasov, P., Wilkins, E., Ann. Biomed. Eng., 23, 6, 833 (1995). U.S. Pat. No. 5,858,186 describes a urea-based biosensor in which substrate hydrolysis is monitored with a pH electrode. U.S. Pat. Nos. 5,945,343 and 5,958,786 describe enzyme-based polymer sensors which fluoresce in the presence of ammonia, which is enzymatically produced from urea and creatinine respectively. In addition U.S. Pat. No. 4,324,858 describes the utilization of cholinesterase for the colorimetric detection of organophosphorus pesticides and nerve agents. A related patent, U.S. Pat. No. 4,525,704 describes the use of cholinesterases and electrical currents in detecting toxic gases.
Generally, enzymatic biosensors function by one of two methods: (1) the sensing enzyme converts an otherwise undetectable compound into another or series of compounds which can be detected by visual, chemical, or electrical techniques; or (2) the enzyme is inhibited by the presence of the compound of interest and enzyme inhibition is linked to a measurable quantity.
Independent of the method of use, the signals of enzyme-based biosensors are often limited in practical application by the nature of enzyme activity. Only in the case of enzyme substrate detection does the sensor provide a positive response in the presence of target analyte. In other words a noticeable change in the sensor indicates the presence of a target analyte. If the detection of enzyme inhibitors or the detection of substrate deficiency is desired, existing approaches rely on negative response signals, or the absence or reduction of an enzymatic reaction, to indicate the presence of inhibitors or the absence of target compounds.
For example, many commercially available nerve agent sensors are based on the inhibition of cholinesterases. The presence of nerve agents blocks the catalytic side on cholinesterase, disabling its ability to catalyze reactions. Such a sensor is employed by exposing the sensing enzyme (cholinesterase) to a questionable environment. Cholinesterase substrate is later applied. Depending upon the substrate or assay system employed, cholinesterase activity may result in a pH change, color change or fluorescent signal. In each of these negative response systems, a signal change occurs only in the absence of analyte (nerve agents). The initial signal of the sensor is unchanged in the presence of analyte. Kumaran, S., and Morita, M. Talanta, 42, 649 (1995). Campanella, L., Colapicchioni, C., Favero, G., Sammartino, M. P. and Tomassetti, M. Sensors and Actuators B, 33, 25 (1996). Hart, A. L., Collier, W. A., and Janssen, D. Biosensors and Bioelectronics, 12, 545-654 (1997). Cho, Y. A., Lee, H. S., Cha, G. S., and Lee, Y. T. Biosensors and Bioelectronics, 14, 387-390 (1999). Bachmann, T. T., and Schmidt, R. D. Analytica Chimica Acta, 401, 95 (1999). Diaz, A., and Ramos Peinado, M. C. Sensors and Actuators B, 38-39, 426 (1997).
It is very desirable to develop sensors and sensing method through which the non-intuitive nature of negative response sensors can be changed to a more intuitive positive response system.
In general, the present invention provides sensors and methods in which the non-intuitive nature of a previously negative response sensor is changed to a more intuitive, positive response system. The present invention is well suited for application in enzymatic biosensors and enzymatic biosensing methods.
In one aspect, the present invention provides a sensor for detecting an analyte in an environment including a first reaction system including at least a first enzyme and at least one substrate for the first enzyme. The analyte inhibits the reaction of the substrate catalyzed by the first enzyme (in other words, the analyte inhibits the first enzyme). The sensor further includes at least a second reaction system that reacts to produce a first detectable state when the first enzyme is inhibited. In some embodiments, the reaction of the first reaction system can produce a second detectible state, different from the first detectible state.
In one embodiment, the reaction of the first reaction system (that is, the reaction of the substrate catalyzed by the first enzyme) causes pH to change in a first direction, and the reaction of the second reaction system causes pH to change in a second direction, opposite of the first direction. The first enzyme can, for example, be a hydrolase, which catalyze hydrolysis reactions, typically resulting in a pH change.
The second reaction system can, for example, include a second enzyme and a substrate for the second enzyme. The second reaction system can also involve a non-enzymatic, chemical reaction. In the case that the second reaction system includes a second enzyme, the first enzyme can, for example, be a hydrolase and the second enzyme can, for example, be a different hydrolase.
The first enzyme and/or the second enzyme can, for example, be immobilized in a polymer medium (for example, in a sponge-like polyurethane) or be in solution. Substrates can, for example, be added to the polymer medium in solution or as a powder.
The first detectible state can, for example, be a colorimetric change. As used herein, the phrase xe2x80x9ccolorimetric changexe2x80x9d refers generally to a detectible change in color. The colorimetric change can be detectible with the human eye or with instrumentation as known in the art.
As set forth above, the reaction of the first reaction system can produce a second detectible state that is different from the first detectible state. For example, the first detectible state can arise from the presence of a first pH sensitive dye producing a colorimetric change, and the second detectible state can be a colorimetric change different from the colorimetric change of the first detectible state.
In another embodiment, the reaction of the first reaction system causes a first colorimetric change and the reaction of the second reaction system causes a second colorimetric change. The second colorimetric change is different from the first colorimetric change.
Furthermore, the reaction of the first reaction system can, for example, cause pH to change in a first direction and the reaction of the second reaction system can cause a pH sensitive colorimetric change when the first enzyme is inhibited.
In another aspect, the present invention provides a sensor for detecting an analyte in an environment including a first reaction system including at least a first enzyme or at least one substrate of the first enzyme. In this embodiment, the analyte is a substrate for the first enzyme if the first reaction system includes the first enzyme, or the analyte is the first enzyme if the first reaction system includes a substrate of the first enzyme. The sensor also includes at least a second reaction system that reacts to produce a first detectable state when the analyte is below a certain concentration. The sensor thus provides a positive or detectible response when the analyte is absent or deficient. Once again, the enzymatically catalyzed reaction of the first reaction system can produce a second detectible state, different from the first detectible state.
In one embodiment, the reaction catalyzed by the first enzyme causes pH to change in a first direction and the reaction of the second reaction system causes pH to change in a second direction, opposite of the first direction.
In another embodiment, the first detectible state arises from the presence of a first pH sensitive dye producing a colorimetric change, and the second detectible state is a colorimetric change different from the colorimetric change of the first detectible state.
In still another embodiment, the reaction of the first reaction system causes pH to change in a first direction and the reaction of the second reaction system causes a pH sensitive colorimetric change when the analyte is below a certain concentration.
The present invention also provides a method of detecting an analyte in an environment including the steps of: providing a first reaction system including a first enzyme and a substrate for the first enzyme, the analyte inhibiting the first enzyme; and providing at least a second reaction system that reacts to produce a first detectable state when the first enzyme is inhibited.
In another aspect, the present invention provides a method for detecting an analyte in an environment including the step of: providing a first reaction system including a first enzyme or a substrate of the first enzyme. The analyte is a substrate for the first enzyme if the first reaction system includes the first enzyme. The analyte is the first enzyme if the first reaction system includes a substrate of the first enzyme. The method also includes the step of providing at least a second reaction system that reacts to produce a first detectable state when the analyte is below a certain concentration.
The present invention thus provides sensors and methods to detect the presence of an enzyme inhibitor or a substrate deficiency (or absence) with a positive signal in form of, for example, changing pH or changing color. Change of pH can be visualized by utilizing pH dyes, electrical equipment, electrodes, or special devices. Once again, color changes can be either inside or outside the visible range, detectable by naked eye or optical instruments. The present invention provides sensors for and methods of monitoring the absence of an enzymatic reaction as a result of inhibitor presence or substrate deficiency (or absence) by, for example, combining a sensing enzyme with the use of an additional enzyme/substrate pair or an additional colorimetric chemical reaction.
The sensors and methods of the present invention can be employed with a wide range of sensing enzymes. As discussed above, several preferred embodiments include hydrolase enzymes such as, for example, lipases, phosphatases, amylases, cellulases, proteases, peptidases, ureases, and deaminases. In general, while catalyzing substrate hydrolysis, each of these hydrolase enzymes causes a corresponding signal, which can, for example, be changing pH, the formation of colorimetric products, or a combination of both. In several embodiments, the sensing enzyme(s) are paired with a second enzyme-substrate combination or a colorimetric chemical reaction. The choice of a second reaction can, for example, depend on the hydrolysis product(s) of the first enzyme in the case of a hydrolase. For example, to compensate the production of hydroxyl or hydronium ions by the first or sensing enzyme, the second reaction can yield hydronium or hydroxyl ions, respectively. In the absence of first enzyme activity, the second reaction produces an excess of either hydroxyl or hydronium ions resulting in a detectible change of pH.
The first or sensing enzyme is not limited to hydrolases, however. In that regard, other classes of enzymes including, but not limited to, oxidoreductases and transferases are suitable using, for example, the formation of colorimetric products. For example, the enzyme peroxidase in combination with the colorimetric substrate dianisidine is suitable to indicate the presence of peroxide.
To compensate the production of colorimetric products by the sensing enzyme, a second reaction can, for example yield a different color capable of changing the overall sensor signal to a third color. In the absence of sensing enzyme activity, the sensor signal indicates the color of this second reaction. For example if a sensing enzyme reaction results in a blue product, a second reaction that yields a yellow product can be used. Both reactions combined yield a green color, whereas the sensor produces only yellow color in the absence of sensing enzyme activity.
While the utilization of enzymes in sensing applications has become commonplace, the sensors and methods of the present invention dramatically improve the signal of such biosensors. There are countless imaginable sensing applications wherein the sensor analyte generates no or reduced enzyme activity. In applications such as sensing of enzyme inhibitors or the deficiency of target compounds, a signal is commonly not achievable with the existing biosensor technology. By definition there is either no or reduced enzyme activity in the presence of inhibitors or the deficiency of the target compound. Turning such negative responses into a much more informative and intuitive positive response is a substantial improvement in the art.
Although the sensors and methods of the present invention are well suited for use in connection with enzymatic reaction systems, the same principles also apply to non-enzymatic reaction systems. Thus, in a further aspect, the present invention provides sensor for detecting an analyte in an environment including a first reaction system that is inhibited (that is, rendered unreactive or reduced in reactivity) by the presence of the analyte. The first reaction can, for example, include two compounds (or one or more compounds and a non-enzymatic catalyst) that react in the absence of the analyte, but the reaction thereof is limited or prevented by the presence of the analyte. For example, the analyte can be a poison for a catalyst present in the first reaction system. The sensor also includes at least a second reaction system that reacts to produce a first detectable state when the first reaction system is inhibited.
The present invention also provides a sensor for detecting an analyte in an environment including a first reaction system including a first compound that reacts with the analyte and at least a second reaction system that reacts to produce a first detectable state when the analyte is below a certain concentration.
In another aspect, the present invention provides a method of detecting an analyte in an environment including the steps of: providing a first reaction system that is inhibited by the presence of the analyte; and providing at least a second reaction system that reacts to produce a first detectable state when the first reaction system is inhibited.
In still another aspect, the present invention provides a method for detecting an analyte in an environment including the steps of: providing a first reaction system including a first compound, the analyte reacting with the first compound; and providing at least a second reaction system that reacts to produce a first detectable state when the analyte is below a certain concentration.